.

Microscopy



1.    INTRODUCTION

2.    EQUIPMENT
3.    IMAGE PROCESSING WORKSTATIONS
4.    FACILITY
5.    SERVICES OFFERED
6.    RATES
7.    BIBLIOGRAPHY
8.    EDUCATIONAL WEB PAGES




LIGHT MICROSCOPY FACILITY (LMF)



1. INTRODUCTION


Researchers at the Andalusian Center for Developmental Biology (CABD) use light microscopy as a tool to study development of cells, organs and embryos in different model organisms. Most of the experimental systems under analysis display complex 3D organisation both at the cellular and organismal levels. Thus, access to the appropriate microscopic techniques to unveil this complexity becomes of paramount importance. Moreover, because of the properties of growing animals, it is necessary to follow their development in real time, which adds further analytical challenges.

3D and 4D (3D + time) Fluorescence Microscopy implies the acquisition of optical sections that need processing to render informative 3D reconstructions. An important characteristic of traditional wide-field microscopy is that each optical section provides both in focus signal, and out of focus emission that needs to be digitally treated to improve the final quality of the image.

The LMF at CABD offers three different solutions to avoid or reduce this unwanted effect:

      1.    Optical Solutions: the use of Confocal Microscopy
      2.    Computacional Solutions: Deconvolution or Image Restoration of widefield collections of images
      3.    A combination of both: Grid Projection

Image Restoration
The word deconvolution refers to the inversion of the distortion introduced with the use of an optical widefield system to acquire fluorescent images. Deconvolution algorithms are based on mathematical models of image formation to subtract or reassign out of focus signal. One of the most optimal methods is the reassignment of each out of focus photon to its original point, a technique termed image restoration.

Confocal Microscopy
Confocal Microscopy is based on sample scanning utilising a point shaped, monochromatic laser beam, also known as a laser line. The light emitted from the specimen is focused by a very small opening, called pinhole (diaphragm) located before the photodetectors. This pinhole cuts away all out of focus information, and allows only the signal from the focal plane to pass through and thus to contribute to image formation.

Grid Projection
A grid of stripes of defined width is projected onto the focal plane of the objective and shifted laterally in three defined steps relative to the sample. A CCD camera takes a picture in each grid position. The three "raw" images are combined into a resultant image by on-line computation. The resultant image is an optical section through the sample, from which all image slices originate. Free of artifacts, with out-of-focus information removed. This image has an improved signal-to-noise ratio, and approximately doubles resolution in axial (Z) direction.


2. EQUIPMENT


The Light Microscopy Facility at the CABD is equipped with the following instruments:

       1.    Stereo Microscope Olympus SZX16

              •    Objectives: 1x and 1,6x with zoom factor up to 11.5 times
              •    Filter Configuration:
                         GFPA BP460-495 BA 510-550
                         RFP 1 BP530-550 BA 575IF
              •    Transmitted Brightfield Illumination Base
              •    Software: CellA



    2.    Confocal Microscope Leica SP2-AOBS.

          •    Vertical Microscope DMRE
          •    Objectives: 10x air, 20x air, 40x oil, 63x oil, 100x oil
          •    Laser lines: 458, 476, 488, 496, 514, 561, 633
          •    Detectors: 3 fluorescence detectors with spectral      
               detection and 1 transmission detector for DIC imaging
   

3.    Confocal Microscope Leica SPE.

     •    Vertical Microscope DM 2500
     •    Objetives: 10x air, 20x multi-immersion, 40x oil, 63x oil
     •    Detectors: 1 fluorescence detector with spectral detection
     •    Laser lines: 405, 488, 532, 635


4.    Restoration Microscopy System DeltaVision

              •    Inverted Microscope Olympus IX71
              •    Objetives: 10x air, 20x air, 40x oil, 60x oil, 100x oil
              •    Camera: CoolSnap HQ
              •    Enviromental Control: Temperature and CO2                         •    Filter Configuration:           
 
 
Flurochromes Excitation Emission
DAPI 360/40 457/50
CFP 436/10 465/30
FITC/GFP 470/40 528/38
YFP 492/18 535/30
DsRed 555/28 617/73
Cy5 640/20 685/40










 


              •    Brightfield illumination
              •    Differential Interference Contrast
              •    Microinjection System for adherent and suspended cells
              •    Software: Softworx
                       •    Z sectioning
                        •    Deconvolution algorithms
                        •    FRAP (laser 488)
                        •    Time Lapse
                        •    Point Visiting
                        •    Panels




     5.    Fluorescence Microscopy Zeiss Axio Imager. M2

              •    Upright Microscope
              •    Objectives: 10x air, 20x air, 40x air, 63x oil, 100x oil
              •    Camera: AxioCam MRm
              •    Filter Configuration:     
 
 
Fluorochromes Excitation Emission
DAPI 365 445/50
GFP 470/40 525/50
Rhodamin 546/12 575-640
Cy5 640/30 690/50
            
              •    Apotome
              •    Brightfield illumination
              •    Differential Interference Contrast
              •    Software: AxioVision 4.7










 

            



     6.    Confocal Microscope Nikon A1R

          •    Inverted Microscope Nikon Eclipse Ti E
          •    Objectives

         
10x  PlanApo  Air  DIC L
20x  PlanFluor  MultiImm DIC N2
30x  PlanFluor Oil  DIC H N2
40x  PlanApo VC Oil DIC N2

              •    Laser Lines 405LD, MultiAr (457, 488, 514), 561DPSS,
                    594, 640LD, 488 (200mW)
              •    Hybrid Scanner: Resonant and non-resonant
              •    32-channel spectral detector 400 to 750 nm
              •    1 transmission detector for DIC imaging
              •    4-channel detector with filters
              •    Emission Filters: 450/50, 482/35, 525/50, 540/30,
                    515/30, 595/50, 585/50, 700/75
              •    Z-movement: motorized stage and piezo
              •    Enviromental Control: Temperature and CO2
             










 

                         

     7.    Spinning Disk Roper Scientific

              •    Inverted Microscope Olympus IX-81
              •    Yokogawa CSU-X1
              •    Objectives: 10x air, 20x air, 40x air, 63x oil, 100x oil
              •    Laser Lines: 405, 491, 561
              •    Emission Filters:  
 
 
DAPI 460/50
GFP 480/40
GFP 525/50
YFP 535/30
RFP 630/75
            
              •    Camera: CoolSnap HQ2 and Evolve
              •    Brightfield and Nomarsky
              •    Temperature control
              •    Bioptech FCS2 Perfussion System
              •    Objective Heater
              •    FRAP: FRAP on the Fly, FRAP Live Reply and FRAP
                    Multidimensional
              •    Software: Metamorph
             















 

        

8.    Confocal Microscope Leica SP5 MP- AOBS

              •    Inverted Microscope DMI 6000
              •    Objetives: 10x air, 20x multi-immersion, 25x water,
                    40x oil, 63x oil, 100x oil
              •    Detectors: 2 PMT and 2 Hybrid GaAsP with
                    spectral detection
              •    Galvo and Resonant Scanner
              •    Laser lines: 405, Ar multiline, 561, 594, 633   
              •    Multiphoton Laser Mai Tai Deep See 690-1040nm   
              •    2 external non-descanned detectors (NDDs)   
              •    Chamber Temperature Controller   
 










 


 
9.    P-30 Vertical Micropipette Puller

              •    Designed for the fabrication of basic micropipettes
                    and patch-type pipettes.
              •    Pulls micropipettes with tip diameters as small as
                    0.3um and moderate taper lengths (10 to 15mm).
              •    Generates pipettes or needles suitable for
                   microinjection studies
    
 
 










 



          
3. IMAGE PROCESSING WORKSTATIONS

   1.   Softworx
   2.   Imaris, ImageJ, LASAF







 

4. FACILITY 


    Contact with Katherina García
    Telephone: 34-95 4977432
    E-mail: kgargar(at)upo(dot)es


5. SERVICES OFFERED:

     - Training of researchers in the use of the instruments and acquisition softwares
     - Training in fundamentals of fluorescence microscopy
     - Training in fundamentals of confocal microscopy
     - Training in fundamentals of image restoration

      For external user: Use of the instruments and acquisition softwares (see rates)
      Contact previously with Katherina García


6. RATES:

System (price per hour) CSIC/UPO OPI Private
Leica SP2 10 € 15 € 20 €
Leica SPE 10 € 15 € 20 €
Spinning Disk 10 € 15 € 20 €
Nikon AIR 10 € 15 € 20 €
DeltaVision 3 € 4,5 € 6 €
Zeiss Apotome 2 € 3 € 4 €


7. BIBLIOGRAPHY

1.    Fluorescence microscopy (1998) Herman, Brian
       Oxford: BIOS Scientific Publishers, cop.
       (535 HER flu)

2.    Introduction to light microscopy (1998) Bradbury, Savile
       Oxford (U.K.): Bios Scientific Publishers; New York: Springer
       (577 IN)

3.    Confocal microscopy: methods and protocols (1999)Totowa, N.J.
       Humana Press, cop.
       (57 CON)

4.    Imaging Living Cells (1999) Rosario Rizzuto. Cristina Fasolato.
       Berlin: Springer
       (576 IMA)

5.    In situ hybridization in light microscopy (2001) Gérard Boca Ratón
       (Florida): CRC Press, cop. 2001
       (577 MOR in)

6.    Methods in cellular imaging (2001)
       Oxford: Oxford University Press
       (57 MET)

7.    Cell biological applications of confocal microscopy (2002)
       San Diego: Academic Press, cop.
       (576 CELL)

8.    Confocal Microscopy for Biologists (2004) Hibbs, A.R.
       (57 HIB con)

9.    Live cell imaging: a laboratory manual (2005)
       Cold Spring Harbor (New York): Cold Spring Harbor Laboratory Press, cop.
       (576 LIV)

10.    Cell Imaging (2006) D. Stephens.
       (576 CELL)

11.    Handbook of Biological Confocal Microscopy (2006) Pawley, J. 
       Plenum
       (57HAN)


8. EDUCATIONAL WEB PAGES


1. Invitrogen: Fluorescence Tutorials
www.invitrogen.com/site/us/en/home/support/Tutorials.html

2. Invitrogen: Spectrum Viewer
http://probes.invitrogen.com/servlets/spectraviewer

3. BD Biosciences: Spectrum Viewer
www.bdbiosciences.com/colors/fluorescence_spectrum_viewer

5. Molecular Expressions Optical Microscopy Primer
http://microscopy.fsu.edu/primer/index.html

6. Nikon: Microscopy Education
www.microscopyu.com/

7. Olympus: Microscopy Resource Center
www.olympusmicro.com/

8. Scientific Volume Imaging: Deconvolution
www.svi.nl/

9. Advanced Light Microscopy Facility. EMBL.
www.embl-heidelberg.de/almf/

10. European Advanced Microscopy Network
www.embl.de/eamnet/index.html

11. Spanish Microscopy Society
http://microscopia.org/

12. European Light Microscopy Iniciative
www.embl.de/eamnet/body